This invention relates to a packing which contains a charged (co)polymer and makes it possible to change the effective charge density or hydrophilic/hydrophobic balance on the surface of a stationary phase in an aqueous system by an external signal (for example, temperature), and a novel separation method by which substances such as metal elements, drugs or biological components are chromatographically separated by using the packing.
There a great variety of liquid chromatography techniques depending on the combination of stationary phase with mobile phase and the interaction systems employed for the separation. Liquid chromatography is a highly important technique for separating metal elements, isolating and purifying drugs and separating peptides, proteins, nucleic acids, etc. in the field of biochemistry. In recent years, moreover, attempts have been made to apply recombinant proteins, etc. produced by bioengineering procedures, which have made remarkable advances, to medicines. Under these circumstances, there is a growing requirement for efficient separation methods for separating and purifying these products. Chromatographic techniques commonly employed at present involve ion-exchange chromatography, reversed phase chromatography, etc.
In ion-exchange chromatography, separation is carried out by using, as a stationary phase, an electrolyte on the surface of an insoluble carrier and irreversibly adsorbing counter ions contained in the mobile phase. As the carrier, silica, cellulose, dextran, styrene/divinylbenzene copolymer, etc. are widely employed. Carriers having ion-exchange groups (for example, sulfonate, quaternary ammonium) introduced thereinto are commercially available as ion exchangers. Solute dissociate into cations, anions and amphoteric ions depending on the hydrogen ion concentration in the solution. When this solution is passed through an ion-exchange column, each ion binds to the oppositely charged exchange group on the carrier surface competitively with solvent ions, thus causing distribution between the solution and the ion exchanger surface at a certain ratio. The migration rates through the column vary depending on the bond strength and separation is completed by utilizing this difference in the migration rate. The distribution can be modified by some methods. For example, it can be changed by controlling the concentration of the competitive ion species in the mobile phase. Alternatively, the extent of ionization of the ion-exchange group on the carrier surface may be varied by changing the hydrogen ion concentration in the solution. That is to say, it has been a practice in ion-exchange chromatography to separate solutes from each other by controlling the ionic strength or the hydrogen ion concentration in the mobile phase to thereby change the elution order of the solutes.
Reversed phase chromatography involves the use of a combination of a hydrophobic stationary phase and a polar mobile phase. Solutes are distributed between the mobile phase and the stationary phase depending on the degree of hydrophobicity. In this case, solutes are eluted also by changing the degree of hydrophobicity of the solvent in the mobile phase to thereby change the distribution between the mobile phase and the stationary phase. Since an organic solvent is employed as the solvent in the mobile phase, it is feared that the activities of the biological components to be separated might be caused to deteriorated thereby.
In short, solutes are eluted and separated from each other fundamentally by varying the solvent in the mobile phase both in ion-exchange chromatography and reversed phase chromatography. Accordingly, there is a risk that the activity of the target sample might be damaged by an acid or organic solvent employed in the elution.
When it is intended to separate substances from each other by two or more chromatographies, each chromatography should be independently carried out, since chromatographic mode varies from carrier to carrier. If it is possible to perform ion-exchange chromatography and reversed phase chromatography by using a single carrier and a single physical stimulus, separation could be completed at an elevated efficiency within a shorter period of time. Moreover, substances which cannot be separated from each other by the conventional techniques can be separated thereby.
There are a great variety of biological components including charged-ones and uncharged ones. In general, a compound capable of being ionized is retained, in an unionized state, in a hydrophobic packing owing to hydrophobic interaction. When ionized, however, the hydrophobic interaction with the hydrophobic packing is weakened. Ion-dissociatable compounds differing in the dissociation constant can be easily separated from each other owing to the ion-ion interaction with the use of an ion exchanger.
It is generally known that weakly acidic ion exchange resins and weakly basic ion exchange resins are suitable respectively for separating basic proteins and acidic proteins. It is thus expected that, by introducing ion-exchange substituents, ion-exchange chromatography based on ion-ion interactions becomes usable in separating various substances, which are similar to each other in hydrophobicity or molecular weight and thus cannot be separated exclusively by hydrophobic interactions, and biological molecules such as proteins and nucleic acid oligomers.
However, there has been known hitherto neither any carrier which is usable both in ion-exchange chromatography and reversed phase chromatography when employed alone under one physical stimulus nor one usable in efficiently separating various substances, which are similar to each other in hydrophobicity or molecular weight and thus cannot be separated exclusively by hydrophobic interactions, and biological molecules such as proteins and nucleic acid oligomers.
To solve the above-mentioned problems, the present inventors have conducted studies and developments from various viewpoints. As a result, they have successfully prepared a novel packing having ion-exchange function by copolymerizing poly(N-isopropylacrylamide)(PIPAAm) with positively charged dimethylaminopropylacrylamide (DMAPAAm) and found that this packing is usable both in reversed phase chromatography and ion-exchange chromatography, when temperature is properly controlled. They have furthermore found that use of the charged copolymer makes it possible to control the LCST of the polymer by regulating pH value. The present invention has been completed based on these findings.
The present invention relates to a method for separating substances characterized by chromatographically separating said substances with the use of a packing which contains a charged (co)polymer and makes it possible to change the effective charge density on the surface of a stationary phase by an external stimulus while fixing a mobile phase to an aqueous system.
The present invention further relates to a method for separating substances characterized by retaining the substances in a stationary phase made of a chromatographic packing chemically modified with a polyalkylacrylamide copolymer having amino, carboxyl, hydroxyl groups, etc., then changing the hydrophilic/hydrophobic balance on the surface of the stationary phase by the temperature gradient method wherein the external temperature is changed stepwise, and passing the substances through a single mobile phase to thereby separate the same.
The present invention furthermore relates to a chromatographic packing which contains a charged (co)polymer and makes it possible to change the effective charge density on the surface of a stationary phase by a physical stimulus.
In the chromatographic packing of the present invention, the charged state of ion-exchange groups on the surface of a carrier can be reversibly controlled by changing the surface structure of the stationary phase by an external physical stimulus such as a change in temperature. Namely, the present invention provides a stationary phase which makes it possible to perform two chromatographic modes, i.e., ion-exchange chromatography and reversed phase chromatography, at the same time with the use of a mobile phase which is a single aqueous solvent (aqueous mobile phase). Moreover, the present invention provides a carrier capable of arbitrarily controlling the charge of ion-exchange groups on the surface of the carrier (in the case of ion-exchange chromatography) or the hydrophilic/hydrophobic balance (in the case of the reversed phase chromatography). The term xe2x80x9caqueous solventxe2x80x9d as used herein means water alone or aqueous solutions containing inorganic salts but free from any organic solvent.
The present invention provides a carrier for separation and purification characterized in that separation is performed by controlling the charge of ion-exchange groups on the surf ace of the stationary phase by regulating the physical properties or structure around the ion exchange groups on the carrier surface by a physical stimulus, while fixing the mobile phase to an aqueous system. According to the present invention, when the external temperature is lower than the critical temperature, the ion-exchange groups appear on the surface of the carrier. Then the biological components to be separated undergo interaction with the ion-exchange groups followed by separation by the ion-exchange chromatography mode. When the external temperature is higher than the critical temperature, on the other hand, the surface charge is weakened and the carrier becomes more hydrophobic. Then, the biological components can be separated by the reversed phase chromatography mode. That is to say, the hydrophilic/hydrophobic balance on the surface of the carrier can be reversibly and arbitrarily changed by controlling the external temperature.